1. Influenza: Biology and Current Prophylaxis and Therapeutics
Influenza A virus is one of the most prevalent respiratory tract infections in humans (Michaelis et al. 2009). Twenty to fifty million U.S. citizens are infected annually resulting in 40,000 deaths. In addition, respiratory virus infections, including flu, are considered to be the leading cause of exacerbation of asthma (Cohen and Castro 2003). For these reasons, the flu virus is considered a significant health threat and a top priority biodefense pathogen by the CDC and National Institutes of Health (NIH). The high risk associated with natural and artificial (bioweapon) flu infection is derived from a number of sources including (1) the ease by which the virus is distributed via aerosol, (2) its ability to escape protective immunity by frequent changes in viral antigens (antigenic drift) and (3) the periodic emergence of new virulent strains resulting from the reassortment of RNA segments between viruses from two different species (antigenic shift). Antigenic drift is the virologic basis for seasonal flu epidemics, while antigenic shift is theorized to be the source of pandemic infections.
Recently, the World Health Organization (WHO) has reported a disturbing increase in the incidence of H5N1 avian influenza infections in human populations. Since 1995, multiple countries have reported outbreaks of H5N1 (Balicer et al. 2007; Uyeki 2009). While the majority of documented cases involve direct viral transfer from wildfowl to human, the WHO and CDC are sufficiently concerned about potential human-to-human transmission to have issued repeated alerts of a H5N1 pandemic. In April 2009, a so-called swine-origin influenza A (H1N1) virus (2009 H1N1 or S-OIV), was identified in Mexico (Dawood et al. 2009). This viral strain originates from triple-reassortant swine flu and is readily transmitted between humans. After its discovery, 2009 H1N1 rapidly spread throughout the world within few weeks. From April to October 17, it was estimated by CDC that 14 million to 34 million infectious cases, 63,000 to 153,000 hospitalizations, and 2,500 to 6,000 2009 H1N1-related deaths occurred. In addition, the epidemiological data indicates that this disease primarily affects people younger than 65 years old, very different from seasonal influenza. Although the death rate caused by 2009 H1N1 was only about 0.45%, lower than the 1918 pandemic (an estimate of 2.5%) and avian flu outbreaks (about 60%), multiple concerns exist. First, mutations in the virus may lead to a more pathogenic strain that increases mortality. Second, since the more deadly avian flu (H5N1; >62%) has also been demonstrated in swine and humans, it is possible that recombination between the 2 strains in either species may result in a highly pathogenic and deadly strain that can migrate readily between species. Third, 2009 H1N1 may reassort with the seasonal flu strains that are resistant to some of the antivirals. This will result in significant difficulties in treating the infected patients.
Currently, two strategies, vaccines and small molecule therapeutics, are utilized to control the spread of flu. Vaccines are predominantly developed from killed or cold-adapted viruses and take advantage of the host immune system to provide limited immunity. While reductions in flu related illness and deaths can be attributed to this approach, especially among high-risk individuals, there are several reasons why vaccines offer limited protection from pandemics. First, the most commonly developed vaccines are based on inactivated viruses. These reagents induce only weak immunity that provides protection for brief (6 month) periods. In light of the facts that 1) only 60-80% of the immunocompetent population and 30-40% of individuals with chronic respiratory ailments (e.g. COPD, asthma) or compromised immune systems receive sufficient protection from vaccination (Kunisaki and Janoff 2009), and 2) vaccines fail to provide protection if administered after infection, alternative approaches are needed to adequately protect the population. A second shortcoming of vaccines involves the complex relationship that exists between the rapid evolution of flu and the limitations associated with reformulating and producing sufficient quantities of vaccines for large populations. The constant evolution of viral antigens frequently renders the previous year's vaccine ineffective and places significant reliance on epidemiological/surveillance data to accurately predict future circulating strains. While these approaches are frequently adequate, the flu outbreak of 2003 and the pandemic in 2009, where predictive models failed to accurately forecast the circulating serotypes and vaccine production capabilities were incapable of responding in a timely manner, demonstrate the need for alternative technologies that can rapidly counter newly emerging serotypes.
In addition to vaccines, there are currently four antiviral drugs approved by the U.S. Food and Drug Administration (US FDA). Two of these agents, amantadine and rimantadine, target the viral ion channel protein, M2. The remaining drugs, zanamivir and oseltamivir, inhibit the function of neuraminidase (NA). While both classes of compounds effectively reduce the severity and duration of flu infections, their efficacy is only limited to the first 24-48 hours after the development of symptoms and can often be associated with side effects. Of still greater concern is the emergence of stable and transmissible drug resistant flu strains (Griffiths 2009; Schirmer and Holodniy 2009). Though the frequency at which NA-targeting drug resistance arises can vary considerably and the associated virulence of resistant strains is often attenuated, exceptions (e.g. the E116G mutation) have been documented (Lackenby et al. 2008). Resistance to oseltamivir has also been identified in the 2009 pandemic virus (www.who.int). Thus, development of a new and flexible anti-flu therapeutic platform is essential.
2. siRNA: A Flexible Molecular Platform for Influenza Prophylaxis and Therapeutics
RNA interference (RNAi) is a naturally occurring, highly specific mode of gene regulation. The mechanics of RNAi are both exquisite and highly discriminating (Siomi and Siomi 2009). At the onset, short (19-25 bp) double stranded RNA sequences (referred to as short interfering RNAs, siRNAs) associate with the cytoplasmically localized RNA Interference Silencing Complex (RISC) (Jinek et al. 2009). The resultant complex then searches messenger RNAs (mRNAs) for complementary sequences, eventually degrading (and/or attenuating translation of) these transcripts. Scientists have co-opted the endogenous RNAi machinery to advance a wide range of basic studies. As siRNAs can be designed to target virtually any gene and can be introduced into cells by a variety of methods, RNAi, represents a highly flexible platform by which researchers and clinicians can control diseases including viral infectious diseases. RNAi has been employed to target a wide range of human pathogenic viruses, including flu and other emerging respiratory infectious viruses (Ge et al. 2003 and 2004; Tompkins et al. 2004; Li et al. 2005, Zhou et al. 2007; McSwiggen and Seth 2008; Zhou et al. 2008; Cheng et al. 2009; Sui et al. 2009). The ability of RNAi to 1) efficiently limit viral replication without reliance on host immune functions, and 2) target multiple genes and/or sequences simultaneously, makes this an ideal therapeutic approach for pathogens that have rapidly evolving genomes (e.g. flu). In particular, siRNA therapeutics may benefit specific patient populations such as infants or the elderly who do not produce a strong immune response to a vaccine and may not have full protection from such a program.
3. Challenges Associated with siRNA Therapeutic Development—Resistant Viral Mutants
The mutation rate of the influenza virus is estimated to be 1.5×10-5 per nucleotide per infection cycle (Parvin et al. 1986). This high frequency of mutation is the underlying basis behind the emergence of variants (escapers) that are resistant to current M2- and NA-targeting chemotherapeutics (Lackenby et al. 2008; Colman 2009). Though RNAi mediated gene knockdown exhibits a degree of resilience to changes in the siRNA target sequence, mismatches in key positions (including nucleotides localized to the siRNA sequence (positions 2-7 of the antisense strand) and target cleavage (positions 9-11) regions can significantly disrupt siRNA efficacy. Multiple studies in which single siRNAs have been developed to target viral pathogens (e.g. HIV, polio virus, Hepatitis B and C viruses) have documented the emergence of escapers. In most of these cases, resistant virus contains one or more changes in the sequence of the siRNA target site (Gitlin et al. 2005; Grimm et al. 2006; von Eije et al. 2008). Mutations in the sequence outside of the siRNA target site were also found in HIV resistant mutants (Leonard et al. 2008). The changes in the RNA secondary structure or/and an evolutionary tuning of viral transcriptional regulation may serve as evasion mechanisms. As escaper populations appear rapidly, novel strategies must be identified if effective RNAi-based viral therapeutics are to become a reality.
Therapeutic strategies that may prevent and/or reduce the emergence of resistant variants include: (1) targeting regions of essential (viral) genes that are highly conserved, and (2) targeting two or more viral genes simultaneously. Moreover, as the rate at which two resistance inducing mutations occur is the product of the frequency of either mutation occurring separately (i.e. 2.25×10-10 per infection cycle), multigene targeting is expected to lessen the frequency at which resistant viral pathogens appear (Gitlin et al. 2005; Grimm et al. 2006; von Eije et al. 2008). We predict that strategies that incorporate both approaches: multiple-gene targeting of conserved regions, will yield the most effective therapeutic platform to combat influenza by RNAi.
4. Challenges Associated with siRNA Therapeutic Development-Delivery Barriers
There are many biological barriers and factors that protect the lungs from foreign particles, such as a thick and elastic mucus layer that may bind inhaled drugs and remove them via mucus clearance mechanism, low basal and stimulated rates of endocytosis on the apical surfaces of well-differentiated airway epithelial cells, the presence of RNase extra- and intra-cellularly, and the presence of endosomal degradation systems in the target cells, among others. Overcoming the difficulties concerning respiratory tract delivery and effective cellular entry and function will pave the way for siRNA as a pulmonary flu therapeutic. In addition, the delivery vehicle and mode of administration must be chosen to be appropriate to the stage of the infection and provide the fastest onset of silencing at the required site of action, e.g. early stage infection/prophylaxis at the lungs and later stage disease through systemic administration. Among them, pulmonary delivery through inhalation provides a noninvasive means for site specific administration to different regions of the lung—resulting in increased bioavailability and fewer adverse effects than with intravenous administration. In a pandemic setting this delivery approach will allow ease of administration directly by patients. In addition, the findings that flu virus is capable of infecting vascular endothelial cells and that endothelial cells express both human and avian flu receptors have urged the development of vascular endothelial cell-directed delivery of antiviral to control some of the systemic syndromes caused by the virus (Sumikoshi et al. 2008; Yao et al. 2008).
Intranasal (i.n.) delivery of siRNA in a mouse model system has successfully reduced expression of a number of endogenous pulmonary targets. Similarly, i.n. delivery of siRNAs targeting a range of viral pathogens (including flu, SARS, and RSV) have successfully knocked down viral gene expression and diminished viral titers (Ge et al. 2004; Bitko et al. 2005; Li et al. 2005). Some delivery carriers were also shown to deliver siRNAs to the mouse lungs via intravenous injection (Ge et al. 2004). In summary, development of formulations for pulmonary delivery of siRNA should be further advanced.